Method of selectively generating fluorescence and its subsequent detection

ABSTRACT

A system for storage and retrieval of information comprising the use of a non-fluorescent material capable of photoconversion to a fluorescent material under light of a particular wavelength and which can be made to fluoresce, after such conversion, by stimulating light radiation of a longer wavelength. A fluorescent image may be formed and detected by the system.

United States Patent [191 [111 3,801,782 Dorion Apr. 2', 1974 METHOD OF SELECTIVELY GENERATING Inventor:

FLUORESCENCE AND ITS SUBSEQUENT DETECTION 1 George Henry Dorion, Riverside, Calif.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: Jan. 27, 1972 Appl. No.: 221,470

Related U.S. Application Data Continuation of Ser. No. 848,578, Aug. 8, 1969, abandoned, which is a continuation-in-part of Ser. No. 764,312, Oct. 1, 1968, abandoned.

U.S. Cl 250/271, 250/461, 250/484 Int. Cl. G0ln 21/00 Field of Search 204/158; 250/483, 484,

[56] References Cited UNITED STATES PATENTS 3,418,470 12/1968 Birkeland 250/71 3,473,027 10/1969 Freeman et al 250/71 Primary ExaminerArchie R. Borchelt Attorney, Agent, or Firm-Charles J. Fickey [57] ABSTRACT 9 Claims, 1 Drawing Figure PLOT 0F OPT/CAL DENSITY 0F PRODUCT A7 430 my TIME OF IRRAD/AT/OA/ 0F /.02 x /0-3M EXAMPLE /3 //v CH2 0/ w/rH 3/3011! LIGHT 0 1 l l 1 l r (/000 SECS.)

1 METHOD OF SELECTIVELY GENERATING FLUORESCENCE AND ITS SUBSEQUENT DETECTION This application is a continuation of application Ser. No. 848,578, filed Aug. 8, 1969, which was a continuation-impart of application Ser. No. 764,312, filed Oct.

1, 1968, both are now abandoned.

This invention relates to storage and retrieval of information by radiation and visual techniques. It more particularly relates to a system for forming a fluorescent image in a non-fluorescent material by radiation with light which converts the material to a fluorescent form and detecting the image with radiation of a different wavelength which does not convert the original compound, but which causes the fluorescent image form to emit light.

Information storage and recovery systems are of rapidly increasing importance in the present-day economy in view of the exponential rise in the number and complexity of the data which must be recorded and be retrievable to handle the increasing every-day business load, and to assist in scientific developments. Many optical systems, including those based on silver halide emulsions and the like, have contributed significantly to this development, largely because of the high packing density with good retrievable resolution inherent in such systems. Systems based on magnetic means, e.g., the well-known magnetic tape and magnetic ink checkprinting systems, have likewise found great utility, largely because of the relative ease of handling and the relatively simple equipment involved, combined with, particularly in the case of the tapes, high reproducible fidelity. However, the optical systems are not as versatile as desired in that only a single image is normally recorded at any one bit, e.g;, the developed Ag image. The same is true of the magnetic tape images where normally only the magnetic image is obtained at any one bit. This image can be made visual by a separate step, e.g., by dusting with iron powder. The magnetic ink images have the advantage of being both visually and magnetically sensible; however, these images suffer from a relatively low packing densiy.

A system for storing and retrieving information has now been discovered which comprises the following: a non-fluorescent material X capable of photoconv ersion to a fluorescent material Y, wherein Y has an absorption band of wavelength longer than the longest wavelength band of X, is irradiated with an information containing beam of light from an ordinary light source or a laser, etc. of such a wavelength to convert the image portion of material X to the fluorescent material Y. A longer wavelength stimulating light radiation is then applied to cause compound Y to fluoresce and thus display the image which can be detected visually or by suitable instrument. Thus material X may be considered as a fluorescer precursor. The radiant source may be of various types providing ultra-violet or infraradiation including lamps, electric arcs, or ultra-violet and infra-red lasers. The image can be formed in any well known manner as by focusing a radiant beam, projecting a beam through a stencil, by use of moving mirror systems with lasers, and the like.

The time required for imaging will depend on the intensity of the radiation source, but is as low as nanoseconds to milliseconds to obtain detectable fluorescence emission.

It will be understood that information formed may be of any desired type, that is alphanumeric characters, code markings such as dots or lines, or pictorial information.

In the present invention, storage of information is rapid, accurate and dry, no fixing being required. Retrieval is rapid, exceptionally sensitive and accurate and is not accompanied by degradation. The inventive technique combines optical deposition of information, causing photochemical reaction in irradiated areas, thus allowing the fine resolution, with detection by fluorescence more sensitive than absorption). As mentioned, no fixing is required where the fluorescer precursor is only sensitive to light of wavelength of less than the detecting beam of radiation. Moreover, it is possible to obtain high information density packing with the present invention.

The inventive method may be used to replace any process that employs change of optical density to change an electrical signal. This may include electronic storage and replay of sound and pictures, numerical data collection and retrieval, and the like; and to produce and validate cards, stamps, passes, and the like documents.

The fluorescer precursor X may be a colorless material, as may also the converted material Y. In this instance, the storage and retrieval may be unknown to all persons except those intended to have knowledge of the information storage. This could be used for placing information on passes or documents to be retained by one person and checked or authenticated by another such as in the case of a gate pass. An advantage of the present system is that any portion or entire cards or documents can be treated with fluorescer precursor material X, even over other information or images, after which particular information may be put on the treated part by light projection. It will be apparent therefore, that many cards may be produced, with individual information placed thereon at a later time, by

conversion of the desired image portion to a fluorescent compound Y. Since the compounds are colorless in either state, space'is saved in that the later information is printed over the original visible information. Detection is preferably, as previously indicated, by means of a longer wavelength light which stimulates the fluorescent form of the material. This stimulating radiation is of such -a wavelength that it does not convert any of the remaining material X to material Y. Although this is the preferable mode of operation in order to prevent conversion of the background to the same state as the image by the detecting radiation, where the detection is relatively of short duration, this could be done by the same wavelength radiation as used to form the image.

The present material has a further characteristic that the amount of detectable fluorescence is proportional to the amount of latent fluorescer which has been converted to the fluorescent state. The amount converted on any radiated area depends on the duration of time of exposure to the irradiating energy. The longer the time period is, the more latent fluorescer there will be converted per unit of exposed area and thus the more intense the fluorescence upon subsequent radiation and detection. This characteristic makes it possible to produce detectable variable tone fluorescent radiation over a given area. This is much like the tone variation in a photographic negative or a magnetic sound tape.

Thus the present invention could be used to prepare a sound tape by audio modulation of the radiant source,

The sound is detectable by conventional fluorescent detection means coupled to audio output means by a suitable transducer. A sound track could be put on a movie film in the same manner, either beside the picture, or printed directly on the film. A phonograph disc could be prepared and played by the same principal.

Although the intensity of fluorescence increases with exposure to radiation, detectable radiation has been obtained with a low intensity light source at as low as one millisecond and with a laser, imaging may be done in nanoseconds. I J

While the converted fluorescent information cannot be removed, it would be possible to insert new information by blotting an old word or number by converting it entirely to a fluorescent bar, and creating another word or number adjacent thereto in fluorescer precursor material X. This, of course, is limited to the area of treatment with fluorescer precursor material X.

The fluorescer precursor material X may be coated on any desired substrate, or it may be incorporated in transparent or opaque plastic films. The substrate may be of any configuration, i.e., sheets, belts, discs, drums, three dimensional objects such as bottles, boxes, and the like. Techniques for this will be readily apparent to persons skilled in the art. It will be obvious that choice of materials may depend on the particular intended use.

The invention may be better understood by reference to the FIGURE. The FIGURE demonstrates the effect of increased exposure of compound X to short wave ultraviolet radiation on the fluorescent emission of product Y under longwave ultraviolet radiation. The FIG- URE is in the form of a graph with the vertical axis representing optical density of product Y, i.e. fluorescence emission, under radiation at 430mp.., and the horizontal axis representing time of irradiation of compound X at 313mg. The specific material X irradiated and converted that of subsequent Example 13, namely o-chlorophenyl-l-naphthyl ether which is nonfluorescent, which upon irradiation with 3 l 3mg. light is converted to an' entirely different chemical compound, benzo [b]-[2,1-d] furan which fluoresces under 340mp. light.

The fluorescer precursor material X may be any material which has the desired properties, that is it has a non-fluorescent form which absorbs light to be converted to a form in which it fluoresces under light of a longer wavelength than that used for conversion to the fluorescent form.

For this purpose, suitable compounds are as follows: 1) 2- 2 f uryl or thienyl) -3-acylc hrornones ofthgjg: mula:

in which R is phenyl, lower alkylphenyl, lower alkoxyphenyl or furyl, Xis a hydrogen or lower alkyl, Y is hydrogen, lower alkyl, lower alkoxy or furoyloxy and Z is oxygen or sulfur. The 2-thienyl compounds such as 2-(2-thienyl)-3-benzoylchromone, 2(5-metl l-2 4 thienyl)-3-benzoylchromone and 2-(5-methyl-2- thienyl)- 3-anisoylchromone are included within the scope of the invention because their photolysismechanism upon exposure to ultraviolet light is substantially the same as that of the 2-( 2-furyl)-3-acylchromones.

The preparation of these chromones is described in greater detail in copending, commonly assigned application Ser. No. 859,607, filed Aug. 8. 1969, which is a continuation-in-part of application Ser. No. 764,294,

F. Feigl, H. E. 'Feigl and D. Goldstein, JACS 77, 4162 (1955); B. N. Mattoo, Trans. Faraday Soc., 52, 1184 (1956); 53, 760 (1957); P. Schwarze and' R. Hoffschmidt, Naturwiss, 46, 205 (1959); C. E. Wheelock, JACS 81, 1348 (1959) COgNa hv/dye sens/H O HO O OH hv/EtOH leuoo-uranlne (dihydrofluoresceln) non-fluorescent COgNa OM/Q uranine (fluoresceln) qsnl uqz rus K. Uchida, S. Kato and M. Koizumi, Bull. Chem. Soc. Japan, 35, 16 1962); K. Uchida and M. Koizumi, ibid., 35, 1871, 1875 (1962); K. Uchida, ibid. 35, 1097 (1963) I b hv 1:! A

non-fluorescent fluorescent W.A. Henderson and A. Zweig, .lACS 89, 6778 (1967) EXAMPLE2 .I ,l E @..CH. 0 uli in EXAMPLE 1 3-Benzoyl-2-(2-furyl) chromone An intermediate compound, Z-furfuryIidene-l-(ohydroxy-phenyl)-3-phenyl-l,3-propanedione is first prepared by condensing o-hydroxy-dibenzoylmethane with furfural. The intermediate may then be oxidized directly to the final product with selenium dioxide. The

reactions are:

o-benzoyloxyacetophenone as described in Organic- Syntheses, Vol. 32 (1952) page 74. A solution of 0.01 gram mole of this material, 0.01 1 gram mole of furfural and several drops of piperidine in 50 ml. of ethanol was refluxed for about 1 hour. The product which separated upon cooling was recrystallized from ethanol. Calculated fOl' C2OH1404; C, H,

FoundE C, 75.49; 1 1, 1.45 u

A mixture of 320 mg. of this material and 220 mg. of

selenium dioxide in ml. of dioxane was refluxed for 6 hours, filtered, and the filtrate evaporated to dryness. The resulting solid was recrystallized from ethanol to five 310 mg. (97 percent yield) of 3-benzoyl-2-( 2- furyl) chromone melting at 2l4.52l5C. v This product is bot h colorless and non-fluorescent. However, it is rearranged into a yellow colored compound exhibiting intense green fluorescence when its dilute solutions in methylene chloride are irradiated with 250-380 millimicron light.

The intermediate compound trans-3-(p-anis oyD-2- (2-furyl)-4-chromanone is first. prepared and is oxidized with selenium dioxide.

A solution in ethanol of 0.01 gram mole of ohydroxy-p'-methoxydibenzoylmethane (W. Baker and- F. Glockling, J. Chem. Soc., 1950, p. 2761) and 0.011 gram mole of furfural was refluxed for 1 hour and cooled and the product was recrystallized from a methanol-water mixture. A 59 percent yield of tan crystals was obtained melting at l36.5-I40C. Analysis: Calculated for O l-L 0 C, 72.40 H, 4.63

Found: C, 72.39; H, 4.64

330 Mg. of selenium dioxide was stirred into a solution of 480 mg. of this product in 15 ml. of dioxane and the mixture was refluxed for 6 hours..The product 3-(panisoyl)-2-(2-fur yl) chromone, was obtained in 83 percent yield. Recrystallization from ethanol afforded cream colored crystals with -a double melting point of EXAMPLE 3 Photolysis of 3-(p-anisoyl)-2-(2-furly) chromone A solution of 16 mg. of the product of Example 2 in 900 ml. of methylene chloride in a quartz vessel was flushed with nitrogen for 30 minutes, stoppered, and irradiated in a reactor equipped with 16 bulbs having a peak light emission at 313 millimicrons. After 1.25 hours irradiation the solution was concentrated in vacuo and combined with the product solutions of other similar runs. The combined solutions were evaporated to near dryness, taken up in a 9:] mixture of petroleum ether and ether, and chromatographed over a silica gel column. This was eluted with petroleum ether-ether mixtures containing progressively more diethyl ether. A series of intermediate fractions were combined, the solvent evaporated, and the residue was crystallized from a methylene chloride-cyclohexane mixture. It was a yellow-orange solid that fluoresced strongly in ultra-violet light. Analysis showed it to be a substituted furo-(3,4-b) chromone of the formula:

I d ,Q-Ocm HC=GCH H II M E EXAMPLE 4 This Example shows the method for condensation of aldehydes with o-hydroxydibenzoylmethanes. In the general procedure, Z-furfurylidenel ohydroxyphenyl)-3-phenyl-1 ,3-propanedione; trans-3 (p-anisoyl)-2-( 2-furyl)-4-chromanone; trans-3-( 3 ,4,5- trimethoxybenzoyl )-2-( Z-furyl )-4-chromanone; and trans-3-(p-anisoyl)-2- chromanone were prepared following the method of (5-methyl-2-furyl)-4- V Baker and Glockling, J. Chem. Soc., 2579 (1962). This is the method used in Examples 40, 4 b, 4c, and 4d. A solution of 0.010 mole of are 6- hydroxydibenzoylmethane, 0.01 1 mole of the aldehyde, and several drops of piperidine in 50 ml of ethanol was refluxed for 30 minutes to 2 hours. The product which separated upon cooling was recrystallized from ethanol.

EXAMPLE 4a Z-Furfurylidenel o-Hydroxyphenyl)-3-phenyll ,3-

' propanedione Reaction of o-hydroxydibenzoylmethane with furfural afforded an 80 percent yield of 4a, mp l54.5159;

Anal. Calcd for C H O C, 75.46; H, 4.43

Found: C, 75.49; H, 4.45

EXAMPLE 4b Trans-3-(p-Anisoyl)-2-(2-furyl)-4-chromanone o-Hydroxy-p-methoxydibenzoylmethane and furfural gave this compound in 59 percent yield, mp l271- 30. Recrystallization from methanol-water afforded tan crystals, mp l36.5-l40; umax 5.97 6.03 p. The nmr spectrum showed the aliphatic ring protons as a pair of doublets centered at 73.94 and 74.72 (.l=l0.5 Hz). Anal. Calcd for C ,H, O C, 72.40; H, 4.63

Found: C, 72.39; H, 4.64

EXAMPLE 4c EXAM PLE 4d Trans-3-(p-Anisoyl)-2-(5-methyl-2-furyl)-4- chromanone This compound was obtained from o-hydroxy-p'- methoxydibe nzoylmethane and S-methyfurfural in 81 percent yield; mp l34-136.5; )tmax 5.92, 6.04 11.. The analytical sample separated as pale yellow needles, mp 137-140. Anal. Calcd for C H O C, 72.92; H, 5.01

Found. C, 72.98; H, 4.94

EXAMPLE 5 3-(3,4,S-Trimethoxybenzoyl)-2-(2-furyl)chromonone Oxidation of the compound of Example 4c afforded a 94 percent yield of 3-(3,4,5-trimethoxybenzoyl)-2- (2-furyl)chromone, mp l60-l6l.5. The analytical sample was obtained as colorless crystals, mp l6l-l6- 2, from ethanol. Anal. Calcd for C H, O-,: C, 67.97; H, 4.46

Found: C, 68.14; H, 4.47

EXAMPLE 6 3-(p-Anisoyl)-2-(5-methyl-2-furyl)chromone A mixture of 1.81 g of the compound of Example 4d and 0.61 g of selenium dioxide in 40 ml of dioxane was refluxed for 6 hours to give 1.51 g (84%) of 3-(panisoyl)-2-(5-methyl-2 furyl)chromone,

Anal. Calcd for C H O C, 73.32; H, 4.48

Found: C, 73.25; H, 4.35

EXAMPLE 7 3-(2-Furoyl)-2-(2-furyl)chromone A mixture of 0.68 g (0.005 mole) of ohydroxyacetophenone, 4.95 g (0.024 mole) of 2-furoic anhydride, and 1.5 g (0.015 mole) of triethylamine was heated at l180 for 5 hours. The cooled black oil was treated with 1:1 methylene chloride-ether and the insoluble product was filtered. The filtrate was washed twice with 5% Na: CO dried, and evaporated and a second crop was crystallized in the same manner. One recrystallization of the combined crops from ethanol gave 0.83 g (54 percent) of 3-(2-furoyl)-2-(2- furyl)chromone, mp 2l2-2l3. A second recrystallization from methanol using charcoal afforded tan crystals, mp 214215. Anal. Calcd for C H O C, 70.59; H, 3.29

Found: C, 70.73; H, 3.37

EXAMPLE 8 3-(2-Furoyl)-2-( 2-furyl)-7-methoxychromone A mixture of 0.83 g of o-hydroxy-pmethoxyacetophenone, 4.1 g of 2 -furoic anhydride, and 1.5 g of triethylamine was heated at 150 for 6 hours and worked up as in the preceding example. 3- (2-furoyl)-2-(2-furyl)-7-methoxychromone was obtained as a tan solid; 0.36 g (21%), mp 234235. Anal. Calcd for C I-1, 0 C, 67.85; H, 3.60

Found: C, 67.37; H, 3.67

EXAMPLE 9 3-(2-Furoyl)-5-(2-furoyloxy)-2-(2-furyl)chromone A mixture of 0.75 g of 2,6-dihydroxyacetophenone, 5.0 g of 2-furoic anhydride, and 1.5 g of triethylamine was heated at 160 for 4 hours. The resulting black solid mass was triturated with 2:1 methylene chlorideether to give 1.85 g of 3-( 2-furoyl)-5-(2- furoyloxy)-2-(2-furyl)chromone as light brown crystals, mp 252253. Trituration with boiling methanol gave tan crystals of the same mp;).;",.' 5.75, 5.99, 6.12

Anal. Calcd for C l-1, 0 C, 66.35; H, 2.91

Ultraviolet irradiation of dilute solutions in methylene chloride of the chromonesof Examples 1-2 and 5-9 resulted in progressive generation of yellow colors accompanied by intense green fluorescence. This solvent was also substituted by others such as methanol, acetonitrile and benzene with essentially equivalent results. The solutions of the chromones, approximately 5 X 10 M, were irradiated with 240-400 my. light from a 1,000 watt high pressure mercury arc.

EXAMPLE 11 A 10' N solution of 1-o-chlorophenylnaphthalene in benzene was irradiated with a 3,130 A. Hanovia watt lamp with Corning No. 9863 and potassium chromate filter. The irradiated product was fluoranthene which gave a blue fluorescence.

. EXAMPLE l2 o-Chlorophenyl- 1 -naphthyl Ether gen at 150 until the base was dissolved The mixture was cooled, and to it was added 100 g (0.50 mole) of a-bromonaphthalene and L2 g of activated copper bronze (United States Bronze Powder Works C-ll8 bronze powder). The whole was stirred vigorously under nitrogen and slowly heated to 190 at which temperature about 20 ml of water was distilled off. The mixture was further heated at 210 for'2 hours. Workup by extraction with aqueous potassium hydroxide and then water followed by distillation gave 14.7 g (16%) of o-chlorophe'nyl-l-naphthyl ether as a viscous, almost colorless oil, b.p. 138l40/0.04 mm. A small sample was further purified by g.l.c. for analysis. Anal. Calcd for C H oClzC, 75.46; H, 4.35; mol. wt. 254.

Foundz C, 75. 83; H, 4.41; m/e 254 EXAMPLE 13 -Chlorophenyl-l-naphthyl Ether' ether as obtained in Example 12, in 300 ml of benzene, was bubbled with nitrogen in a quartz vessel. The solution was then irradiated with 254 my. light in a Rayonet reactor for 18 hours. The resulting yellow solution was I evaporated and the residue chromatographed on alumina. The appropriate fractions (110 mg. 65%) were combined, sublimed, and recrystallized 3 times from hexane and once from methanol to give 55 mg (32%) of benzo[b]naphtho-[2,l-d]furan, mp 102-103 (Liter. 103 This compound gave a blue fluorescence.

Determination of Yield and Quantum Yield A solut qwf 25-5 na istsmw lether in ml of benzene was irradiated in a l g I1!J-lltra' violet quartz cell with 313 my. light of known intensity. The rate of formation of benzo[b]-[2,l-d]furan was followed by observing the appearance of its long wavelength peak at 340 my The quantum yield calculated therefrom was 3.2 X 10*.

A portion of the above solution diluted to H100 was irradiated in the same cell for 18 hours with 300 my. light. The yield of benzo[b]-2[2,l-d]furan calculated from the size of the 340 my. peak was 79 percent.

EXAMPLE 14 ple 3. The rate of product formation versus time of ex-- posure is illustrated by the FIGURE. A detectable fluorescence is present after less than a second and the den-- sity increases with the time of exposure Thus the storage of data in analog form is readily accomplished by suitably modulating the irradiating light.

The irradiating light can be modulated for a duration of exposure over different portions of the area treated with the fluorescer precursor compound to obtain information in analog form. A similar result could be obtained by modulating the irradiating light intensity over the treated area. The information is detechable witha suitable photocell coupled to a demodulation means to interpret the data.

While certain specific examples and preferred modes of operation have been set forth, it will be obvious that this is solely for illustration, and that various changes and modifications may be made in the invention without departing from the spirit of the disclosure and the scope of the appended claims.

I claim:

l. A method for processing information which comprises irradiating with an information containing beam of ultraviolet light not exceeding about 4,000 angstroms in wavelength, a substrate incorporating a fluorescer precursor compound which is non-fluorescent in its initial state, said compound beingconverted under said ultraviolet light of said information containing beam to a form in which it will fluoresce under stimulating untraviolet light, thereafter irradiating said converted compound with said stimulating light, and detecting the fluorescent emission.

2. The method of claim 1 wherein said fluorescer precursor compound is 2-( 2-furyl)-3-acylchromone.

3. The method of claim 1 wherein said fluorescer precursor compound is a 2-(2-thienyl)-3-acylchromone.

4. The method of claim 1 wherein said stimulating light has a wavelength greater than said converting given wavelength.

5. The method of claim 1 wherein said fluorescer precursor is coated on a substrate.

6. The method of claim 1 wherein said fluorescer precursor is incorporated in a film which is the substrate.

7. The method of claim 1 wherein said fluorescer precursor is present as a coating on a substrate over information on said substrate detectable by visible light.

8. The method of claim 1 wherein said fluorescer precursor is applied to said substrate as the initial step of the imageing process. v

9. The method of claim 1 wherein the intensity of said given wavelength light is varied to vary the fluorescence emission under said stimulating light. 

1. A method for processing information which comprises irradiating with an information containing beam of ultraviolet light not exceeding about 4,000 angstroms in wavelength, a substrate incorporating a fluorescer precursor compound which is non-fluorescent in its initial state, said compound being converted under said ultraviolet light of said information containing beam to a form in which it will fluoresce under stimulating untraviolet light, thereafter irradiating said converted compound with said stimulating light, and detecting the fluorescent emission.
 2. The method of claim 1 wherein said fluorescer precursor compound is 2-(2-furyl)-3-acylchromone.
 3. The method of claim 1 wherein said fluorescer precursor compound is a 2-(2-thienyl)-3-acylchromone.
 4. The method of claim 1 wherein said stimulating light has a wavelength greater than said converting given wavelength.
 5. The method of claim 1 wherein said fluorescer precursor is coated on a substrate.
 6. The method of claim 1 wherein said fluorescer precursor is incorporated in a film which is the substrate.
 7. The method of claim 1 wherein said fluorescer precursor is present as a coating on a substrate over information on said substrate detectable by visible light.
 8. The method of claim 1 wherein said fluorescer precursor is applied to said substrate as the initial step of the imageing process.
 9. The method of claim 1 wherein the intensity of said given wavelength light is varied to vary the fluorescence emission under said stimulating light. 